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CYP703 Is an Ancient Cytochrome P450 in Land PlantsCatalyzing in-Chain Hydroxylation of Lauric Acid to ProvideBuilding Blocks for Sporopollenin Synthesis in Pollen W

Marc Morant,a Kirsten Jrgensen,a Hubert Schaller,b Franck Pinot,b Birger Lindberg Mller,a

Danie` le Werck-Reichhart,b and Sren Baka,1

a Plant Biochemistry Laboratory, Department of Plant Biology and Center for Molecular Plant Physiology, Faculty of Life Sciences,

University of Copenhagen, DK-1871 Frederiksberg C, Copenhagen, Denmarkb Departments of Plant Metabolic Responses and Plant Isoprenoids, Botanic Institute, Louis Pasteur University, 67083 Strasbourg

Cedex, France

CYP703 is a cytochrome P450 family specific to land plants. Typically, each plant species contains a single CYP703.

Arabidopsis thaliana CYP703A2is expressed in the anthers of developing flowers. Expression is initiated at the tetrad stage and

restricted to microspores and to the tapetum cell layer. Arabidopsis CYP703A2 knockout lines showed impaired pollen

development and a partial male-sterile phenotype. Scanning electron and transmission electron microscopy of pollen from the

knockout plants showed impaired pollen wall development with absence of exine. The fluorescent layer around the pollen

grains ascribed to the presence of phenylpropanoid units in sporopollenin was absent in the CYP703A2 knockout lines.

Heterologous expression of CYP703A2 in yeast cells demonstrated that CYP703 catalyzes the conversion of medium-chain

saturated fatty acids to the corresponding monohydroxylated fatty acids, with a preferential hydroxylation of lauric acid at the

C-7 position. Incubation of recombinant CYP703 with methanol extracts from developing flowers confirmed that lauric acid and

in-chain hydroxy lauric acids are the in planta substrate and product, respectively. These data demonstrate that in-chain

hydroxy lauric acids are essential building blocks in sporopollenin synthesis and enable the formation of ester and ether

linkages with phenylpropanoid units. This study identifies CYP703 as a P450 family specifically involved in pollen development.

INTRODUCTION

Plants began to colonize land 450 to 600 million years ago. Thetransition from water to land required adaptation to more ex-

treme light, temperature, and water conditions. An essential

prerequisite for this step was the ability to protect the ungermi-

nated gametophyte. A key element in this strategy was the

development of sporopollenin, a biopolymer in spores from

mosses and in pollen. Sporopollenin constitutes the main con-

stituent of exine, the outer layer of spores and pollen. Exine not

only affords a protective barrier against pathogen attack, dehy-

dration, and UV irradiation but also facilitates pollen recognition

and adhesion to the stigma and thus has a role in protection as

well as pollination (Edlund et al., 2004). Although sporopollenin is

an essential contributor to important exine characteristics, the

chemical composition of this polymer remains elusive, as it is

difficult to chemically degrade into defined components and toisolate in large amounts. Studies on the chemical similarities

between the wall of spores and pollen and on the composition of

sporopollenin were first reported in 1937 (Zetzsche et al., 1937).

Recently, based on acidic methanolysis of exine from cattail

(Typha angustifolia), sporopollenin was proposed to be com-

posed of a limited number of different monomers (Bubert et al.,

2002). Polyhydroxylated unbranched aliphatic units as well as

phenolic constituents were identified as the main monomeric

units in different plant taxa (Wehling et al., 1989; Ahlers et al.,

1999, 2000; Bohne et al., 2003). High-resolution solid state 13C

NMR spectroscopy identified oxygen atoms present in ether,

aliphatic and phenolic hydroxy, carboxy, and ester groups

(Guilford et al., 1988; Ahlers et al., 2003). Covalent coupling of

the monomeric units of sporopollenin by ether linkages has been

proposed to provide the characteristic high resistance to chem-

ical degradation (Ahlers et al., 2000). Studies of Arabidopsis

thaliana male-sterile mutants with abnormal exine formation

have shown that tapetal cells as well as the pollen grain con-

tribute to exine biosynthesis (Bedinger, 1992). Analysis of the

male-sterile mutantsmale sterile2(ms2) (Aarts et al., 1997),de-

fective in exine patterning1 (dex1) (Paxson-Sowders et al., 2001),

no exine formation1 (nef1) (Ariizumi et al., 2004), and faceless

pollen1(flp1) (Ariizumi et al., 2003) indicate the incorporation of

fatty acids and the transport of components from the sporophytic

surroundings as integral steps in sporopollenin deposition.

Cytochromes P450 (P450s) are hemethiolate monooxygena-

ses involved in a vast array of biosynthetic pathways in second-

ary and primary metabolism (Werck-Reichhart et al., 2002;

Morant et al., 2003). P450s involved in the synthesis of pivotal

1To whom correspondence should be addressed. E-mail bak@life.

ku.dk; fax 45-35-28-33-33.

The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy described

in the Instructions for Authors (www.plantcell.org) is: Sren Bak

([email protected]).WOnline version contains Web-only data.

www.plantcell.org/cgi/doi/10.1105/tpc.106.045948

The Plant Cell, Vol. 19: 14731487, May 2007, www.plantcell.org 2007 American Society of Plant Biologists


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backbone structures characteristic of different classes of primary

and secondary metabolites are highly conserved throughout the

plant kingdom (e.g., CYP73, the cinnamic acid 4-hydroxylase,

catalyzing the initial step in phenylpropanoid synthesis; and

CYP51, catalyzing the essential 14a-demethylation step in sterol

biosynthesis) (Nelson et al., 2004). Based on phylogenetic stud-ies, plant P450s can be divided into 10 separate clans that cover

the current 61 families (Nelson et al., 2004; Galbraith and Bak,

2005; http://www.p450.kvl.dk/p450.shtml). The demand for the

recruitment of novel catalytic abilities and for enhanced sub-

strate profiles is reflected by a large number of duplication

events. Accordingly, most P450 families in higher plants are

proliferated andcontain multiple paralogs. As an example of this,

273 DNA sequences encoding P450s have been identified in the

fully sequenced Arabidopsis genome (http://www.p450.kvl.dk/

p450.shtml). Based on the biochemistry catalyzed by known

P450s from the 10 different clans, plant P450s can been divided

into three major groups. The first diverse and ancient group in-

cludes P450s with a function that preceded the colonization of

land by plants. This group is extantin Chlamydomonas reinhardtiiand contains clans 51 and 710, involved in sterol biosynthesis;

clan 97, involved in the biosynthesis of carotenoids; and clan

711, the function of which is unknown. The second group in-

cludesP450sthat evolved as a consequenceof thedevelopment

of multicellular terrestrial life forms, as represented by clan 85,

involved in the biosynthesis of hormones for signaling and de-

velopment (e.g., brassinosteroids and gibberellins); clan 74,

involved in jasmonic acid biosynthesis; and fatty acid monohy-

droxylases in clan 86, required for cutin synthesis. The third

group comprises the highly proliferated clan 71. This clan in-

cludes P450s involved in the biosynthesis of the majority of plant

natural products involved in adaptation to abiotic and biotic

stress (e.g., P450s involved in lignin and flavonoid synthesis).

While most P450 families have proliferated through time, it is

striking that four families (CYP703, CYP715, CYP722, and

CYP724) are represented by single members in Arabidopsisas

well as in Oryza sativa (Nelson et al., 2004). The fact that these

four families exist as discrete and highly conserved single family

members across taxa strongly indicates that they encode an

essential and conserved biological function. Of these four single

family members, a function has been assigned to CYP724.

CYP724 is involved in the early C-22 hydroxylation step in

brassinolide biosynthesis common to all land plants (Ohnishi

et al., 2006). The CYP703 family has been studied in Petunia

hybrida, in which the gene was shown to be expressed in the

early stage of flower bud development. Although recombinant

petunia CYP703A1 was shown to convert lauric acid into un-

known products,the biological function of theCYP703 familyhas

remained elusive (Imaishi et al., 1999).

Here, we show that the CYP703 family is present in all land

plants examined. We demonstrate that Arabidopsis CYP703A2

is expressedduring pollen development. Knockout ofCYP703A2

impairs pollen development, as the exine is not developed, and

results in a male-semisterile phenotype. Heterologous expres-

sion of CYP703A2 identified capric (C10), lauric (C12), and

myristic (C14) acids as saturated fatty acid substrates and

identified the products formed as the corresponding in-chain

monohydroxylated fatty acids preferentially hydroxylated at

carbon atom 7. Methanol extracts from developing flowers

identified lauric acid and in-chain hydroxy lauric acid as the in

planta substrate and product, respectively. This indicates that in-

chain hydroxylated fatty acids are key components in sporopol-

lenin synthesisduring exine formation andidentifies CYP703as a

P450 family specifically involved in pollen development.

RESULTS

The CYP703 Family Evolved with Land Colonization and Is

Conserved among Land Plants

EST databases constitute a powerful resource to survey the

species and tissues in which a gene is expressed. EST data-

bases were mined with Arabidopsis CYP703A2 (At1g01280) as

the query, and 43 putative CYP703 ESTs were identified from 17

different species (Table 1),mainly belonging to angiosperms and

gymnosperms. The overrepresentation in angiosperms corre-

sponds to the larger number of angiosperm ESTs present in

public plant databases. Based on the available data, CYP703appears to be a single-family gene in seed plants. The recent

availability of the genome of the moss Physcomitrella patens

(http://moss.nibb.ac.jp/cgi-bin/blast-assemble) allowed for a

search for CYP703 orthologs in nonvascular plants. Three puta-

tive CYP703 orthologs were identified in the moss. Full-length

coding sequences were identified by contig assembly, and

homology with the CYP703 family was confirmed by phyloge-

netic analyses (Figure 1). According to the general nomenclature

rules, P450s are assigned to thesame family when the sequence

identity at the amino acid level is >50%. The three moss se-

quences show


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CYP703A2, which spans the entire promoter, were fused to the

b-glucuronidase (GUS) reporter gene. Sixteen independent

PCYP703A2:GUS lines were selected and analyzed in both the

Wassilewskija and Columbia (Col-0) ecotypes. GUS staining was

performed at different stages of plant development (stages 1, 3,

and 6, according to Boyes et al. [2001]). All transgenic lines

examined displayed the same distinct expression pattern and

revealedthat expression was restricted to anthers (Figure 3B). To

more precisely determine the expression in anthers, plastic-

embedded GUS-stained flower sections were analyzed. The

tapetum cell layer andthe microspores were identified as the cell

types expressing CYP703A2. Expression was initiated at the

tetrad stage (Figure 3G). During pollen maturation, the tapetum

cells started to degenerate as their function was completed and

CYP703A2expression vanished (Figures 3I and 3J).

CYP703A2 Knockout Mutants Have Reduced Fertility

Two T-DNA insertion lines in the Arabidopsis Col-0 ecotype,

SALK_119582 (Alonso et al., 2003) and SLAT N56842 of

At1g01280, were identified. The T-DNA insertions were mapped

to exons 1 and2, respectively(seeSupplemental Figure1 online).

In both T-DNA insertion lines, the absence ofAt1g01280 tran-

scripts in developing flowers was verified by RT-PCR (data not

shown). Both T-DNA insertion lines displayed the same visual

phenotype, characterized by slightly longer inflorescences, an

extended period of blooming, a reduced number of elongated

siliques, and overall reduced seed set. Analysis of the primary

inflorescences at maturity showed that 31 6 7% (n 12 plants)

ofthe siliquesfailed todevelop in the SLATN56842 lineand 306

6% (n 10 plants) failed to develop in the SALK_119582 line.

By contrast, only 6 6 3% (n 11 plants) of siliques failed to

develop in Col-0 wild-type plants. The impaired siliques were

mainly located at the base of the inflorescences, while the more

apical siliques appeared similar to wild-type siliques (Figure 4A).

Two backcrosses using wild-type pollen confirmed that the

phenotype observed is linked to the absence of CYP703A2. In

both CYP703A2 knockout lines, the anthers within the first

flowers formed as the lower part of the inflorescence wilted

and were supported by a very short filament (data not shown). In

the middle part of the inflorescence, filaments were longer than

within the flowers positioned below but still shorter than in the

wild type, and pollen release was not observed (cf. Figures 4B

and 4D). In apical flowers, the amount of pollen was reduced

Table 1. Occurrence of Putative CYP703 Orthologs in EST and Genomic Databases

Taxonomic Group Species Tissues Used for Library Accession Number

Green algae Chlamydomonas reinhardtii http://www.chlamy.org No hits

Bryophytes Physcomitrella patens http://moss.nibb.ac.jp/

cgi-bin/blast-assemble

Three contigs

Gymnosperms Ginkgo biloba Gingko mi crospo rophyll CB094452, CB09 4744

Picea glauca Male strobili developmental

sequence

CK442130, CK442743, CK442745

Pinus taeda Tripl Ex polle n cone lib rary AW754540

Amborella trichopoda Early male flower bud CV012183

Angiosperms

Monocots

Aegilops speltoides Premeiotic anther BQ841303

Oryza sativa Panicle (>10 cm) C99519, AU094669, AU064265a

Secale cereale Ant her before ant hesis BE 495344, B F145314, CD453339,

BE495003, BE494625, BE493881

Triticum aestivum Spike at meiosis BJ211945, BJ218997, BJ219398

Zea mays Mixed stages of anther and pollen AW506768

Eudicots

Arabidopsis thaliana Flower bud AV535036, AV532990b

Mixed tissues (including flower) AA720028

Lotus corniculatus Flower bud BP040316, BP034275c

Populus trichocarpa Male catkin V050E11

Antirrhinum majus Whole plant (including flower) AJ805079, AJ793628, AJ808979

Ipomoea nil Flower and flower bud BJ567452, BJ574200, B J558599,

BJ554486

Lactuca sativa Mixed tissues BQ994698, BU001758

Solanum lycopersicum 3- to 8-mm flower bud BI929405

8 mm to preanthesis bud BI931896, BI932066, BI932102

Solanum tuberosum Flower bud CV506033, CV502932, CV504543

The different entries represent putative Arabidopsis CYP703A2 orthologs that were identified in EST databases encompassing algae, bryophytes,

gymnosperms, monocots, and eudicots. The strong bias for expression in flowers buds, male organs, and tissues before anthesis is apparent.

Analyses at the genome level in C. reinhardtiiand P. patens are shown at top.aThese ESTs correspond to O. sativa CYP703A3.bThese ESTs correspond to Arabidopsis CYP703A2.cThese ESTs correspond with 99.6% identity to L. japonicus CYP703A7.

CYP703, a P450 in Exine Formation 1475


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compared with the wild type, whereas filament length appeared

similar to that of the wild type (cf. Figures 4A and 4C).

Scanning electron microscopy of pollen from middle and

apical flowers revealed that the surface of the pollen from the

mutant was smooth while the surface of wild-type pollen ex-

hibited the characteristic ridged surface (Figures 4A to 4D).

Cross-pollination of theCYP703A2insertion lines with wild-type

pollen fully restored seed production, whereas wild-type plants

crossed with pollen from theinsertion lines resulted in a very poor

seed production. This confirmed that the phenotype was asso-

ciated with pollen, that pollen production was impeded, and that

at least some of the pollen released was able to germinate. In

agreement with this, pollen from the insertional lines germinated

in vitro under high humidity (data not shown). The reduced seed

production in the two CYP703A2 knockout lines most likely

reflected the combination of a reduced amount of mature pollen,

Figure 1. Phylogenetic Tree Displaying the Position of the CYP703 Family within the P450 71 Clan.

Bootstrap values are shown at the nodes. The CYP703 family is highlighted in gray. At,Arabidopsis thaliana; Bs,Berberis stolonifera; Gm,Glycine max;

Lj, Lotus japonicus; Me, Manihot esculenta; Mt, Medicago truncatula; Ph, Petunia hybrida; Pp, Physcomitrella patens; Ps, Pisum sativum; Os, Oryzasativa; Sb,Sorghum bicolor. Sequences marked with asterisks were deduced from genomic data.

1476 The Plant Cell


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a poor pollen survival rate, and reduced filament growth. Only

within the more apical flowers did the combined effects of

increased filament length, slightly increased pollen viability, and

increased pollen numbers serve to improve the chance of the

pollen to come into direct contact with the stigma and thus

enable pollination, despite impaired pollen survival and release

rates (Figure 4C).

Knockout of CYP703A2 Impairs Pollen Wall Architecture

Microscopic observations of open anthers in apical flowers of the

CYP703A2 knockout lines revealed a reduction of the quantity of

mature pollen, as some of the pollen grains were arrested in

development. The pollen grains from anthers at the basal parts of

the inflorescences werehighlydegenerated, and few microspores

went through the entire maturation process. Accordingly, all sub-

sequent pollen observations were based on pollen isolated from

anthers from apical flowers. Mature pollen from wild-type lines as

well as from the two CYP703A2 knockout lines contained two

generativecells and a single vegetativenucleus (Figures 5A to 5C),

indicating that meiosis and mitosis occurred normally.

Microscopic analyses of longitudinal sections of mature an-

thers under UV light revealed that the characteristic fluorescent

layer around thepollen grainsfrom theCYP703A2 knockout lines

was lacking (Figures 5H and 5I). The fluorescence around the

wild-type pollen originates from phenylpropanoid units that are

part of the sporopollenin in the exine layer. The absence of this

fluorescent layer demonstrated that the sporopollenin compo-

nent of the exine layer was structurally altered or absent.

The size and shape of the microspores in the wild type and the

CYP703A2 knockout line were analyzed by scanning electron

microscopy (Figure 5). Whereas wild-type microspores exhibited

a uniform size, microspores from theCYP703A2 knockout line

could easily be divided into two size classes. One class con-

tained microspores that had a similar size as wild-type spores,

andthe other class containedmicrosporesthat were a lotsmaller

(Figure 5F). At thetetrad stage, themicrospore size ofCYP703A2

knockout lines was indistinguishable from that of the wild type

(data not shown). This suggests that the small microspores were

arrested in development after tetrad release (Figure 5F, white

arrows). Analyses of mature pollen grains by scanning electron

microscopy revealed that the characteristic ornamented patternof the surface of the wild-type pollen ascribed to the exine layer

was lacking in the CYP703A2 knockout lines, which instead

exhibited a smooth surface (Figures 5D to 5G). Likewise, the

three characteristic furrows of the Arabidopsistricolpate pollen

were missing in pollen from theCYP703A2 knockout lines. When

Figure 2. Detection of CYP703A2 Transcript in Selected Tissues as

Monitored by RT-PCR.

Tissues from floweringArabidopsisCol-0 plants from stages 6.10 to 6.50

(Boyes et al., 2001) were analyzed for the presence of CYP703A2

transcripts by RT-PCR. After 40 cycles of PCR, CYP703A2 transcript

was detected exclusively in closed flower buds. Actin1(At2g37620) was

used as a control gene, and primers were designed to exclude the

amplification of genomic DNA.

Figure 3. Floral Expression ofCYP703A2as Analyzed by GUS Stainingof Transgenic PCYP703A2:GUS ArabidopsisPlants.

(A) and ( B) Global view of GUS-stained inflorescences in wild-type (A)

and PCYP703A2:GUS(B) plants.

(C) to (J) Sections of plastic-embedded anthers after GUS staining in

wild-type ([C] to [E]) and PCYP703A2:GUS reporter fusion([F] to [J]) plants.

Ta, tapetum layer; Te, tetrad.

(C) and (F) Meiotic stage.

(D) and (G) Tetrad stage.

(E) and (H) After tetrad release.

(I) Tapetum degeneration.

(J) After tapetum degeneration.



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scanning electron microscopy analysis was performed without

prior fixation, pollen from theCYP703A2 knockout mutant lines

collapsed, while wild-type pollen resisted the vacuum applied

during fixation (cf. Figures 5D and 5F with 5E and 5G). The

structural and physical protection provided by the exine layer in

the wild type was thus missing in pollen from the CYP703A2

knockout lines.

In order to analyze the structure of the mutant pollen wall in

further detail, transmission electron microscopy was undertaken

on mature wild-type and knockout pollen grains (Figure 6).

Transmission electron microscopy sections of wild-type pollen

exhibited a white layer corresponding to the intine layer that

covers the plasma membrane of the pollen. Thesubtle dark layer

covering the intine layer corresponds to nexine, the internal part

of the exine layer. The external exine layer is ornamented by

baculum and tectum, two types of structure that appear gray on

the transmission electron microscopy sections. The invagina-

tions within the tectum and baculum layer contain a white-gray

matrix designated tryphine. Tryphine is deposited at the end of

pollen development and completes the formation of the mature

pollen coat. In contrast with the well-organized architecture of

the wild-type pollen wall, the knockout pollen sections revealed

an irregular intine layer and a total absence of the characteristic

exine layer. Theirregularity of the plasma membrane observed in

the knockout lines might be a reflectionof thelacking exine layer.

In addition, small vesicles supposedly targeted with material for

exine formation accumulate in the knockout lines. It is not clear

whether the dark and irregular layer covering the intine corre-

sponds to the nexine layer or a residual deposition of tryphine or

a mixture of both.

CYP703 Catalyzes In-Chain Hydroxylation of Saturated

Medium-Chain Fatty Acids

To characterize the catalytic function of CYP703A2, the enzyme

was heterologously produced in yeast WAT11 cells optimized for

the expression of plant P450s (Pompon et al., 1996). This

expression system has been used as a reference system for

the heterologous production of eukaryotic P450s for the past 10

years. The expression level and folding state of the P450 can be

monitored by measuring the absorption of the reduced complex

between P450 and carbon monoxide (Omura and Sato, 1964).

The WAT11 yeast strain coexpresses the Arabidopsis NADPH

P450 Reductase1 (ATR1), which delivers electrons from NADPHto the P450. Yeast microsomes that contained 53 pmol of

CYP703A2 per milligram for microsomal protein were isolated

and used in an in vitro screen for putative substrates. A range

of fatty acids and phenylpropanoids were tested, as exine is

thought to be composed of fatty acidderived and phenylpro-

panoid-derived monomers (data not shown).

In the presence of NADPH, CYP703A2 was found to catalyze

the in vitro monohydroxylation of saturated medium-chain fatty

acids (C10, C12, C14, and C16), as analyzed by thin layer

chromatography. Lauric acid (C12) was the preferred substrate

(Figure 7). The turnover number of CYP703A2 with lauric acid as

substrate (20 min1) was similar to that of CYP94A1 involved in

cutin monomer synthesis (Tijet et al., 1998). Activity was not

observed toward caprylic acid (saturated C8 fatty acid), stearic

acid (saturated C18 fatty acid), unsaturated fatty acids like

C18:1, C18:3, and C20:4, and phenylpropanoids like trans-

cinnamic acid andp-coumaric acid. Microsomes isolated from

untransformed WAT11 yeast cells did not catalyze the hydrox-

ylation of fatty acids.

The substrate specificity exerted by CYP703A2 was further

analyzed by gas chromatographymass spectrometry (GC-MS).

This substantiated that the C10, C12, and C14 saturated fatty

acids were allmonohydroxylatedat carbon atom 6, 7, or 8 (Figure

7). A minute amount of hydroxylation at carbon 9 was also

observed with lauric acid as substrate. Lauric acid (C12) was

more efficiently hydroxylated compared to capric (C10), myristic

(C14), and palmitic (C16) acids. Independent of the fatty acid

chain length, carbon atom 7 was the major site of hydroxylation

(Figure 7; see Supplemental Figure 2 online).

As in other eukaryotic cells, fatty acids are generally not freely

available in plants but are typically bound as triglycerols or CoA

esters (Browse and Somerville, 1991; Daum et al., 2007). To

identify the in planta substrate and product of CYP703A2,

lyophilized methanol extracts from developing wild-type flowers

were incubated with yeast microsomes containing CYP703A2

and analyzed by liquid chromatographymass spectrometry

(LC-MS). As expected, free lauric acid was detected in neither

the methanol extracts from the flowers nor in the microsomal

Figure 4. Comparison of Inflorescences for Col-0 and a CYP703A2

Knockout Line.

Siliques arrested in development in the main inflorescence are indicated

by white arrows. Dissection of flowers at stage 14 (Smyth et al., 1990)

was done forthetop part([A] and [C]) and the middle part ([B] and [D]) of

primary inflorescences. The combination of shorter filament and reduced

pollen release (D) may explain the observed reduction of seed produc-

tion for the middle part of the inflorescence. The abnormal pollen

structure seen by scanning electron microscopy is conserved in the

middle and top inflorescences (cf. [A] and [B] with [C] and [D]). No

obvious differences were seen in the structure of pollen for the knockout

between the middle and top flowers. In the CYP703A2 knockout lines,

the basal flowers displayed brown or incompletely developed anthers.

1478 The Plant Cell


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membrane preparation (data not shown). However, upon incu-

bation of the methanolic flower extract with yeast microsomes, a

large pool of free lauric acid was released, due to the activity of

yeast triacylglycerol lipases and acetyl-CoA thioesterases that

copurified and adhered to the microsomal membrane prepara-

tion (Figure 8A). In the presence of NADPH, lauric acid liberated

from the flower extract by the yeast microsomes was metabo-

lized by CYP703A2 into four new metabolites with a molecular

mass corresponding to that of monohydroxylated lauric acid

(Figure 8A, gray peaks). The same product profile was obtained

when CYP703A2-containing microsomes were incubated with

free lauric acid (Figure 8B). The GC-MS analyses demonstrate

that the four product peaks correspond to hydroxylation at the

C6, C7, C8, and C9 positions, with the main product (retention

time 21.5 min) representing hydroxylation at the C7 position.

Capric acid and to some extent also myristic acid were identified

as substrates in the in vitro experiments with isolated fatty acids

(Figure 7). Yeast triacylglycerol lipases and acetyl-CoA thioes-

terases are known to exert a broad substrate specificity (Daum

et al., 2007).Accordingly, release of capric or myristic acid would

be expected if the corresponding conjugates were present in the

flower extracts. Neither capric acid nor myristic acid or their

Figure 5. Comparison of Pollen from CYP703A2 Knockout Plants and Wild-Type Pollen.

(A)to(C) TrinucleatedArabidopsispollen in Col-0 (wild-type) (A), SLAT N56842 knockout line(B), and SALK_119582 knockout line (C). Gc, generative

cells; Vn, vegetative nucleus. Nuclei in isolated pollen from wild-type and CYP703A2knockout lines were visualized by staining with 49,6-diamidino-

2-phenylindole dihydrochloride.

(D)to(G) Surface structure ofArabidopsispollen from wild-type plants ([D]and [E]) and theCYP703A2knockout line ([F]and [G]) after fixation of the

tissues ([D] and [F]) or without fixation ([E]and [G]), monitored by scanning electron microscopy. Bars 10mm.

(H) and (I)Autofluorescence under UV irradiation of mature anthers from wild-type (H) and CYP703A2 knockout (I) lines. Autofluorescence is impaired in

the CYP703A2 knockout line due to the lack of exine deposition. ex, site for exine deposition. The two CYP703 knockout lines displayed identical

phenotypes.



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corresponding monohydroxylated products could be detected

when flower extracts were incubated with CYP703A2-containing

yeast microsomes, which strongly indicates that lauric acid and

lauric acid monohydroxylated at the C6, C7, C8, or C9 position

with preferentialhydroxylation at theC7 position are thein planta

substrate and products of CYP703A2 (Figure 8A).

DISCUSSION

We have identified CYP703A2 as a lauric acid in-chain hydrox-

ylase involved in exine formation. Mutants lacking CYP703A2

were male-semisterile and were characterized by a smooth

pollen surface, as opposed to the usual characteristic ornamen-

ted surface (Figures 4 and 5). Several male-sterile mutants have

previously been characterized with reduced or abolished exine

wall. flp1 has an altered pollen surface (Ariizumi et al., 2003).

FLP1 has been suggested to play a role in the synthesis of the

tryphine,the mixture of fatty acidsand proteins embedded within

theexinelayer(Figure 6) andresponsiblefor pollenhydration and

pollenstigma interaction and recognition. In contrast with

CYP703A2, FLP1 is also expressed in stems and siliques. The

nef1 mutant is also impaired in exine development. NEF1 is a

chloroplastic membrane protein that may be involved in either

lipid biosynthesis or lipid transport (Ariizumi et al., 2004), under-pinning the importance of lipids in exine biosynthesis. The

mutantdex1, lacking the exine characteristic pattern, has been

described as defective in a membrane-associated protein

(At3g09090) most likely involved in early exine (primexine) for-

mation (Paxson-Sowders et al., 2001). Based on the phenotype,

expression patterns, and catalytic function of CYP703A2, we

propose to name the twoCYP703A2 knockout mutantsdex2-1

(SLAT N56842) and dex2-2 (SALK_119582), for defective in exine

patterning2.

In angiosperms, male sterility can occur due to dysfunctions in

anther and pollen development (Sanders et al., 1999). Some

mutant lines are male-sterile, while in other mutants, only a

reduction of fertility is observed. Mutants affected in exine

development, like ms2, encoding a protein homologous withfatty acid reductases (Aarts et al., 1997), often exhibit reduced

seed set. Like the CYP703A2 knockout mutant dex2, MS2 is

expressed during pollen maturation and ms2 mutants have

brown and shrivelled anthers, reduced production of pollen

grains, and the majority of the seeds are produced from apical

flowers formed at the inflorescence. Both the ms2 mutant and

the CYP703A2 knockout mutant dex2 characterized in this study

exhibit impaired pollen walls, and the development defect is

observed after the pollen mother cell has undergone meiosis

(Aarts et al., 1997). Thestrong phenotypic resemblance between

these two mutants suggests that both loci could serve key

functions in sporopollenin synthesis and exine production.

Based on these two mutants, we conclude that sporopollenin

deposition during exine formation is important but not essential

for seed production inArabidopsis.

CYP703A2 Is a Lauric Acid in-Chain Hydroxylase

The impaired exine production in theCYP703A2knockout lines

(Figures 4 to 6) and the ability of CYP703A2 to catalyze in-chain

monohydroxylation of medium-chain fatty acids (Figure 7) indi-

cate that CYP703A2 provides medium-chain hydroxy fatty acids

as essential building blocks during sporopollenin synthesis and

exine formation. When in-chain hydroxy lauric acids are not

formed, the fluorescent layer around the pollen ascribed to

sporopollenin is absent. This lack of in-chain hydroxylated lauric

acids prevents the formation of cross-links in the form of ester

and ether linkages between the two key building blocks of

sporopollenin. Methanol extracts from developing flowers re-

vealed the presence of a large pool of conjugated lauric acid that

may serve as an intermediate storage form of lauric acid for

sporopollenin biosynthesis. The absence of capric (C10) and

myristic (C14) esters in flower extracts further argues that lauric

acid (C12) is the in planta substrate of CYP703A2. This is in

agreement with the in vitro screening that identified lauric acid

as the best substrate (Figure 7). Accordingly, in-chain monohy-

droxylated lauric acids are essential building blocks in spo-

ropollenin synthesis and thus are required for proper pollen

Figure 6. Ultrastructure of Pollen Wall from Wild-Type and Insertion Line

SALK_119582 Plants as Seen by Transmission Electron Microscopy.

(A) Transmission electron microscopy sections of wild-type pollen coat

revealed a regular intine layer covered with a nexine layer and an outer

structured sexine layer with the typical ornamented structures com-

posed of bacula and tecta and covered with pollen coat.

(B) By contrast,the exine layer was absent in both insertion lines, as seen

here for the SALK line. Only an irregular intine layer is visible, together

with a layer that might be the nexine layer or the residual pollen coat.

Compared with the wild type, the plasma membrane of the CYP703A2knockout line appears highly irregular, and vesicles supposedly filled

with material targeted for exine formation seem to accumulate in the

cytosol.

bc, baculum; pc, pollen coat; pm, plasma membrane; tc, tectum.

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development and viability. Hydroxy lauric acids possess a hy-

droxy as well as a carboxy group that, together with phenyl-

propanoid units like p-coumaric or caffeic acid (Wehling et al.,

1989), provide oxygen atoms required for the formation of ether

and ester linkages during biosynthesis of the sporopollenin

polymer (Guilford et al., 1988; Ahlers et al., 2003). Ahlers et al.

(2000, 2003) previously suggested the presence of aliphatic units

containing more than one oxygen atom in sporopollenin of

cattail. The documented ability of CYP703A2 to preferentially

hydroxylate carbon atom 7 of lauric acid offers a route to

synthesize building blocks that secure a uniform distance be-

tween the cross-links in sporopollenin. This provides increased

resistance to evaporation by controlling the level of reticulation of

the pollen wall. The presence of additional ether linkages orig-

inating from other hydroxy groups residing in phenylpropanoids

would further augment the strong resistance of the polymer to

chemical degradation.

Independent of fatty acid chain length, CYP703A2 preferen-

tially catalyzed hydroxylation at carbon atom 7 (Figure 7). This

catalytic property is unique for a plant fatty acid hydroxylase, as

most other fatty acid hydroxylases are end chain (v) hydroxy-

lases (Kandel et al., 2006). Accordingly, we propose that in

CYP703A2, the fatty acid carboxy group is positioned deep

within the active site and anchors the fatty acid with the hydro-

phobicchaintightly secured in thebinding pocketof theenzyme,

thus facilitating in-chain hydroxylation independent of fatty acid

chain length, preferentially at the C7 position (Figure 7). A similar

orientation of fatty acids has been observed in CYP152A1 from

Bacillus subtilis, which catalyzes hydroxylation at the a- or

b-carbon (C2 or C3) of long-chain free fatty acids. In CYP152A1,

the carboxy group interacts with the positively charged guanid-

inium group of Arg-242 in the I-helix and thus positions the

substrate so that the a- orb-carbon is in proximity to the heme

within the active site (Lee etal.,2003). By contrast, in the Bacillus

megaterium fatty acid v-1- or v-2-hydroxylase (P450BM-3 or

CYP102), the fatty acid carboxy function is positioned at the

entrance of the substrate access channel and thus serves to

position thesubstrate with thevposition in proximityto theheme

(Ravichandran et al., 1993). A similar orientation of the fatty acid

has been suggested for CYP81B1 from Helianthus tuberosus,

which is an v-1- to v-5-hydroxylase (Cabello-Hurtado et al.,

1998). Future crystal structures or homology modeling studies

Figure 7. CYP703A2-Catalyzed Hydroxylation of Fatty Acids of Different Chain Lengths and Identification of Carbon Atom 7 as the Main Site of

Hydroxylation.

Medium-chain saturated fatty acids were incubated in the presence of 10 pmol of CYP703A2, and products were analyzed by thin layer

chromatography and GC-MS. For hydroxy capric acid and hydroxy lauric acid, the horizontal gray bars represent the relative ratio of hydroxylation

for each carbon. Due to thelow conversion rates, the relative ratios could notbe determinedfor hydroxy myristic acid. For thesame reason, theposition

of the hydroxylation of palmitic acid could not be determined. Three potential hydroxylation sites (?) are proposed based on the observed hydroxylation

patterns for capric, lauric, and myristic acids.



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shouldhelp to elucidatehow lauricacid is positioned in theactive

site of CYP703A2.

CYP703A2 Coexpresses with Genes Involved

in Exine Formation

The formation of exine and the biosynthesis of the sporopollenin

polymer involve multiple genes. To identifyputatively coregulated

genes, the Genevestigator microarray database (Zimmermann

et al., 2004) wasminedusingCYP703A2 as thequery. Two genes

previously known to be involved in pollen development after the

meiotic step,At4g14080 (r2 0.9149) and MS2 (r2 0.816), were

identified. At4g14080encodes a callase involved in degradation

of the callose wall during pollen development (Hird et al., 1993),

and MS2 (At3g11980) encodes the fatty acid reductase that when

mutated gives rise to the male-sterilems2line (Aarts et al., 1997).

According to the model proposed by Dong et al. (2005), degra-

dation of the callosewall of the microspore occurs simultaneously

with the biosynthesis of sporopollenin in primexine and exine.

Thus, the observed coexpression pattern provides independent

support for a key function of CYP703A2 in exine biosynthesis. A

more general investigation of coexpression patterns using Ex-pression Angler (Toufighi et al., 2005) identified several genes in

phenylpropanoid synthesis, including At4g34850, At1g02050

(both encoding a chalcone or stilbene synthase), At1g62940

(encoding a 4-coumarate-CoA ligase), andAt1g68540(encoding

a cinnamoyl-CoA reductaselike protein). In addition,At3g42960

(encoding an alcohol dehydrogenase), At1g75790 (encoding a

multicopper oxidase type I family protein), and CYP704B1

(At1g69500), belonging to clan 86 comprising fatty acid hydrox-

ylases, were identified as being coexpressed with CYP703A2.

This coexpression pattern underpins the concerted synthesis of

fatty acids and phenylpropanoids.

Evolution of CYP703, a Prerequisite for the Development

of Terrestrial Plants

A major challenge encountered by the first land plants was to

protect their spores from dehydration and damage by UV irra-

diation. TheCYP703 familyhas been a keyplayer in this process,

and CYP703A2 is a documented example of a P450 shown

specifically to be involved in pollen development. The high

conservation in terrestrial plants, the specific expression in

developing flowers, the conserved substrate specificity among

petunia and Arabidopsis, and the observed deficiency with

respect to pollen wall development in the T-DNA insertion lines

strongly suggest that theCYP703 familyis exclusively devoted to

a key step in pollen development after the tetrad release stage.

The general expression pattern in male tissues of gymnosperms

and angiosperms (Table 1) documents a general function of the

CYP703A family in pollen development of spermatophytes.

Knockout of allene oxide synthase, CYP74A, has been shown

to confer male-semisterility in Arabidopsis (Park et al., 2002).

However, CYP74A is involved in biosynthesis of the phytohor-

mone jasmonic acid, and the expression of CYP74A is not

restricted to anther or pollen, in agreement with a more general

role in signal transduction pathways (e.g., by wounding).

EST database searches identified CYP703A orthologs in an-

giosperms and gymnosperms (Table 1). When compared, the

ESTs listed in Table 1 indicated the presence of a singleCYP703

transcript inPicea,Secale,Triticum,Antirrhinum,Ipomoea, and

Solanum. In the fully annotated genomes ofArabidopsis and

O. sativa, unique CYP703 orthologswere alsoidentified (Figure 1,

Table 1). Similarly, in Populus trichocarpa, a single functional

sequence,CYP703A4, and a pseudogene were identified. The

recent genome duplication of the poplar genome (Blanc and

Wolfe, 2004) might explain the presence of two alleles in this

particular species, in which one of the alleles has become a

pseudogene while the other paralog has retained the original

function (Walsh, 1995). No orthologs have been identified in

ferns. This may simply reflect the limited amount of sequence

information available within this taxonomic group. In agreement

with a unique function in terrestrial plants, noCYP703orthologs

could be identified in the fully sequenced genome of the green

Figure 8. LC-MS Chromatograms of the Incubation of CYP703A2 with

Flower Extract and Lauric Acid.

(A)Methanol extracts from wild-type Arabidopsisflowers were used for

in vitro incubations with yeast microsomes containing CYP703A2 in the

presence or absence of NADPH. In the presence of NADPH, lauric acid

(black peaks) was converted into four monohydroxy lauric acid products

(gray peaks), as characterized by extracted ion chromatography.

(B)The same product pattern was observed when 10 nmol of lauric acid

was used as a substrate.

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alga C. reinhardtii(http://www.chlamy.org/). C. reinhardtiicon-

stitutes a valid ancestor to land plants (Willis and McElwain,

2002), and the absence of the CYP703 family in this organism

as well as other algae demonstrates that the CYP703 family

appeared after the radiation of land plants from the chlorophyte

group during the adaptation to a terrestrial life form.

In mosses, spores may be regarded as equivalent to pollen in

that they are the result of a meiotic division and are protected by

a similar cell wall composed of sporopollenin (Zetzsche et al.,

1937). In agreement with this, three members of the CYP703

family, designated CYP703B1, CYP703B2, and CYP703B3,

were identified in theP. patensgenome (Figure 1, Table 1). It is

not understood at present why the P. patens genome contains

three apparently fully functional CYP703 paralogs while theother

known genomes contain a single copy. However, divergence be-

tween mosses and angiosperms occurred around 425 million

years ago (Willis and McElwain, 2002), and subsequent duplica-

tion events and neofunctionalization of the CYP703 ancestral

gene could be a valid explanation. Analysis of expression pat-

terns and heterologous expression of the enzymes encoded by

the threeP. patens CYP703 paralogs should reveal whether the

three gene copies are truly redundant or whether they have ac-

quired new functions or become pseudogenes, as observed in

poplar. The possibility to delete genes in P. patens by homolo-

gous recombination should facilitate the clarification of this

issue. We suggest that at least one of the CYP703B alleles in

P. patensis involved in spore wall development.

The need to protect the gametophyte is not restricted to

terrestrial plants. In the salt water alga Chlamydomonas monoica

(Chlorophyta), the zygospore is protected by the biopolymer

algaenan. The algaenan biopolymer is known to contain C22,

C24, and C26 fatty acids and alcohol units linked by ether and

ester linkages (Blokker et al., 1999). In contrast with algaenan,

sporopollenin contains phenylpropanoids that provide the nec-

essary additional protection from UV irradiation and dehydration

(Figure 5). Based on the ability of CYP703A2 to provide prefer-

entially C7 monohydroxylated lauric acid and on the model for

algaenan (Blokker et al., 1999), we propose a model for the

impact of CYP703A2 in sporopollenin biosynthesis (Figure 9).

Monomeric hydroxylated fatty acid units produced by CYP703

and phenylpropanoids like p-coumaric acid and caffeic acid

joined by ether and ester linkages provide the backbone of the

sporopollenin. To provide further strength to the sporopollenin

polymer, additional ether linkages can be envisioned added by

subsequent but currently unknown hydroxylases. Whether some

of the coexpressed genes identified by Genevestigator or

Figure 9. Model of the Role of CYP703A2 in Sporopollenin Formation.

Monomeric units derived from CYP703-catalyzed hydroxylation of lauric acids are shown in red. The participation of oxygen atoms within these units in

ester and ether linkages in the formation of the sporopollenin biopolymer is illustrated by circles and squares, respectively. The p-coumaric and caffeic

acid units illustrate the presence of phenylpropanoids in the sporopollenin polymer.



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Expression Angler encode hydroxylases providing additional

hydroxy groups remains an open question.

The freshwater algae charophytes represent some of the

most primitive organisms hosting the phenylpropanoid pathway

(Kroken et al., 1996). Identification ofCYP703orthologs in fresh-

water algae may provide new knowledge on the origin of spo-ropollenin. The occurrenceof phenylpropanoid units in freshwater

algae also supports the notion that the early phenylpropanoid

pathway evolved before the lignin pathway. Accordingly, the

lignin pathway may have evolved from the sporopollenin path-

way. Thus, it is tempting to speculate that the major determinant

in the evolution of the phenylpropanoid pathway was to provide

monomeric aromatic units that, upon incorporation into sporo-

pollenin freshwater algae spores, provided the necessary resis-

tance to UV irradiation (e.g., when ponds dried out during the

climatic changes that preceded the evolution of terrestrial

plants).

METHODS

CYP703A Expression

Sequence Database Mining

Searches for putative CYP703A2 orthologs were performed using

TBLASTN searches (Altschul et al., 1990) (blosum62 matrix) in the plant

EST database at http://www.ncbi.nlm.nih.gov/ usingArabidopsis thaliana

CYP703A2 as the query sequence. Candidate gene sequences were

analyzed for similarities to the CYP703 family by an additional BLAST

(BLASTX) search in the plant P450 database at http://www.p450.kvl.dk/

blast.html, currently encompassing 573 P450 sequences, including all

ArabidopsisP450 sequences. Multiple sequence alignments and a boot-

strapped neighbor-joining phylogenic tree based on 1000 iterations were

constructed using ClustalX (Thompson et al., 1997) using the default

parameters (Gonnet protein weight matrix series) (Gonnet et al., 1992)

and MEGA 3.1 (Kumar et al., 2004), respectively, and subsequently used

to confirm the assignment to the CYP703 family. The multiple sequence

alignment used to construct the phylogenetic trees can be downloaded

from our website (http://www.p450.kvl.dk/Figure1_Alignment_Marc.pdf).

The Gene Atlas tool at Genevestigator (https://www.genevestigator.

ethz.ch/) was used to determine the expression pattern ofCYP703A2

(At1g01280) in Arabidopsis. The search included data from experiments

performed with wild-type plants and the 22K Arabidopsisdata set. At the

time of the analysis, 1388 array experiments representing 1388 RNA

extractions from callus (6 slides), cell suspensions (71 slides), seedlings

(308 slides), inflorescences (170 slides), rosettes (626 slides), and roots

(207 slides) were included in the 22K data set.

Phylogenetic Analysis

The bootstrapped neighbor-joining tree was built in MEGA 3.1 (Kumar

et al., 2004) with a set of protein sequences representative of the P450s

from clan71. Thetwo non-clan 71 P450s,CYP51A1 andCYP710A1,were

used as an outgroup to root thetree,as they areancient P450s involved in

essential steps in thebiosynthesis of sterols present in all plants and thus

precede the occurrence of the 71 clan. The tree was bootstrapped with

1000 iterations (node cutoff value of 50%). The underlying amino acid

sequences in FASTA format (see Supplemental Data 1 online) and the

multiple alignment (see Supplemental Data 2 online) can be accessed at

http://www.p450.kvl.dk/Review2006/Figure1_Sequence_FASTA.tfa and

http://www.p450.kvl.dk/Figure1_Alignment_Marc.pdf, respectively.

CYP703A2-Promoter GUS Fusion and Plant Transformations

The 1000 nucleotides upstream of the start codon ofCYP703A2 were

amplified from Col-0 genomic DNA using the primers 59-ATACATG-

TAAAGCTTACGAAAGAGAAGCTGATATCGGCCTTGTCTAGAGA-39 and

59-CGAATATGAGGATCCCTAATCTTATATTCTATATTCAACAAGTCTTT-

TGA-39 containing the restriction sites HindIII and BamHI (underlined),respectively. The PCR products obtained were ligated into the binary

vector pBI121 (Clontech). Agrobacterium tumefaciens strain LBA4404

holding the plasmid Ti pAL4404 was transformed with this construct

harboring the NPTII gene conferring kanamycin resistance (Hoekema

et al., 1983). TheArabidopsis lines Wassilewskija and Col-gl1 were trans-

formed by the floral dip method (Clough and Bent, 1998). Transformed

plants were selected on Murashige and Skoog agarplates supplemented

with kanamycin (50 mg/L).

RNA Extractions and RT-PCR

Total RNA was extracted with the RNeasy Plant Mini kit (Qiagen) from

flowers at stages 6.10 to 6.50 (Boyes et al., 2001). RNA (1 mg) was

reverse-transcribed with the iScript cDNA synthesis kit (Bio-Rad), and

CYP703A2 expression was subsequently visualized by PCR with the

primers 59-TTAAAGCTCTAATTCAGGACATGATAG-39 and 59-CCATA-

AACTTCCACCGTATCAATATTTC-39 using the Phusion High-Fidelity

DNA polymerase (Finnzymes) and following the manufacturers protocol.

Forty amplification cycles were performed: 10 s at 988C,30sat 608C,and

15 s at 728C.Actin1(At2g37620) was used as a control with the primers

59-GGTCGTACTACCGGTATTGTGCT-39 and 59-TGACAATTTCACGC-

TCTGCT-39. For bothCYP703A2and Actin1, primers were designed to

be mRNA-specific by spanning an intron.

Characterization of CYP703A2 Knockout Lines

Two T-DNA insertion lines forAt1g01280 in ecotype Col-0, SLAT N56842

and SALK_119582 (alias N619582), were obtained from the Nottingham

Arabidopsis Stock Centre. Genotyping of the lines with respect to the

At1g01280locus was done with the primers 703for2 (5 9-TACCTAGGA-

GATTACTTGCCATTTTGGAG-39) and 703rev2 (59-ACGTCTCTCGTAC-CACACAACGTAGATAG-39). In the SLAT N56842 line, the presence of the

T-DNA insertion was detected with the primers 703for2 and dspm1

(59-CTTATTTCAGTAAGAGTGTGGGGTTTTGG-39) or 703rev2 and dspm11

(59-GGTGCAGCAAAACCCACACTTTTACTTC-39). In the SALK_119582

line, T-DNA insertion in the At1g01280 locus was detected by PCR with

the primers 703rev2 and LBa1 (59-TGGTTCACGTAGTGGGCCATCG-39)

or 703for2 and 59-ACTCTAATTGGATACCGAGGGGAATTTAT-39. PCR

fragments containing T-DNA border sequences were sequenced for

verification of the T-DNA insertion (see Supplemental Figure 1 online).

Histology

Petals and sepals of Arabidopsis flowers were removed by dissection

using a Leica MZ FLIII stereomicroscope fitted with a Leica DC 300F

camera. Anther sections were analyzed using a Leica DMR fluorescencemicroscope, likewise fitted with a Leica DC 300F camera.

Arabidopsisflowers were embedded in plastic according to the man-

ufacturers manual for Technovit 8100 (Heraeus) with minor alterations.

The tissues were dehydrated in a graded series of acetone solutions (25,

50, and 100%, 1 h each) and left overnight in the filtration solution.

Sections (5 mm) were cut on a Reichert-Jung 2030 rotary microtome.

Mature anthers were dissected and pollen grains releasedin a solution of

mannitol (0.3 M) and 49,6-diamidino-2-phenylindole dihydrochloride (0.3

mM) (Fluka). After incubation, pollen grains were analyzed with a Leica

DMR fluorescence microscope.

GUS colorations were performed on freshly harvested plants by a swift

ethanol (70%) wash followed by incubation (378C, 16 h) in GUS staining

1484 The Plant Cell


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solution (Jefferson, 1989). Stained tissues were dehydrated by successive

washes in ethanol solutions of increasing strength (50 to 96%). GUS

sections were counterstained with periodic acid Schiff, starting with an

overnight incubation in dimedon(0.5%). Thefollowing day, sections were

washed in running tap water (15 min) followed by incubation (10 min) in

freshly made periodic acid (1%). Before incubation in Schiff reagent (30

min), the sections were washed (running tap water, 10 min). Directly from

the Schiff reagent, the sections were incubated (3 3 2 min) in Na2S2O5(0.5%). Finally, the sections were rinsed (running tap water, 10 min),

followed by a brief wash (1 min) in ion-exchanged water.

For scanning electron microscopy, dissected flowers were vacuum-

infiltrated with a solution of glutaraldehyde (2.5%)and paraformaldehyde

(2%) in phosphate buffer (0.1 M, pH 7.2). Flowers were washed three

times with a phosphate buffer (0.1 M, pH 7.2), dehydrated in a graded

series of ethanol, critical-point dried (CPD 020; Balzers Union), and

mounted on specimen stubs using double-sided tape. The samples were

coated with gold:palladium (3:2) in an ion sputter (JFC-1100; JEOL) and

analyzed with a scanning electron microscope (LEO 435VP) with sec-

ondary electron detector at high voltage (10 kV). Plants analyzed without

prior fixation were infiltrated with phosphate buffer (0.1 M, pH 7.2).

For transmission electron microscopy analysis, the anthers were

vacuum-infiltrated and fixed with 2.5% glutaraldehyde and 2% parafor-maldehydein 100mM Na-phosphate buffer, pH 7.0, for3 h. Sampleswere

postfixed in 1% osmium tetroxide for 2 h, dehydrated in acid dimethoxy-

propane and acetone, and embedded in Spurr resin (Ted Pella). Ultrathin

sections were stained with uranyl acetate and lead citrate and observed

with a JEM-1010 transmission electron microscope (JEOL).

Heterologous Expression in Yeast

The CYP703A2 cDNA was amplified by PCR from theflower cDNA library

CD4-6 (Weigel et al.,1992)using the primers 59-CGATATTCTTGGATCC-

ATGATTTTGGTGCTAGCCTCC-39 and 59-CGATATTCTTGGTACCTTA-

TGTGTACAAATGAGCTGC-39 harboring the restriction sitesBamHI and

KpnI (underlined)and ligated into pGEM-T(Promega). Theinternal BamHI

site in CYP703A2 was removed by quick-change strategy mutagenesis

(Stratagene). The verified coding sequence was inserted in the multicopyshuttle vector pYeDP60 and subsequently transformed into Saccharo-

myces cerevisiae strain WAT11, engineeredto coexpress ATR1 (Pompon

et al.,1996). Expression of CYP703A2 was induced by galactose (Pompon

et al., 1996), and expression levels were estimated by carbon monoxide

difference spectrometry (Omura and Sato, 1964).

In Vitro Enzyme Assays with Lipid Substrates and Flower Extracts

Microsomes (10 mg protein/mL, 10 pmol of P450) isolated from yeast

expressing CYP703A2 were incubated (30 min) with a series of radiola-

beled fatty acids (100 mM) of different chain lengths in Na-phosphate

buffer (50mM, pH 7.4). After incubation,the reaction mixture wasapplied

toa thin layer chromatographyplate (Silica Gel60 F254sheets; Merck)and

developed in diethyl ether:light petroleum:formic acid (50:50:1, v/v/v)

(Pinot et al., 1998). For GC-MS analysis, products were eluted from thethin layer chromatography plates with diethyl ether:hexane (50:50, v/v; 10

mL), methylated with diazomethane, and silylated with a mixture of

pyridine and N,O-bistrimethylsilyltrifluoroacetamide containing 1% (v/v)

trimethylchlorosilane (1:1, v/v). GC-MS analysis was performed using an

Agilent 6890 series gas chromatograph fitted with a capillary column (0.25

mm3 30 m, 0.25-mm film thickness [HP-5MS]). The gas chromatograph

was combined with a quadrupole mass selective detector (Agilent

5973N). Mass spectra were recorded (70 eV) and analyzed as described

by Eglinton et al. (1968).

Flowers from inflorescences containing flowers at every stage of

development, from small buds to the last stage before silique elongation,

were extracted with hot methanol. Floral tissues (500 mg) were boiled for

5 min in 1 mL of 85% methanol. Twenty microliters of this extract were

lyophilized and subsequently used as substrate for in vitro assays with

CYP703A2, as described above. Control assays were performed with

100mM lauric acid. After 30 min of incubation at 288C, reaction mixtures

were stopped by the addition of ethyl acetate, concentrated in vacuum,

and analyzed by LC-MS.

LC-MS was performed using a HP1100 liquid chromatograph (Agilent

Technologies) coupled to a Bruker Esquire 3000 ion trap mass spec-

trometer (Bruker Daltonics). An XTerra MS C18 column (Waters; 3.5 mm,

2.13 100 mm) was used at a flow rate of 0.2 mL/min. The mobile phases

were A (0.1% [v/v] HCOOH and 50 mM NaCl) and B (0.1% [v/v] HCOOH

and 80% [v/v] MeCN). The gradient program was as follows: 0 to 2 min,

isocratic 18% B; 2 to 30 min, linear gradient 18 to 100% B; 30 to 50 min,

isocratic 100%B. Thespectrometerwas runin negative electrospray mode.

Plant Culture Conditions

Plants were cultivated in an autoclaved mixture of soil:vermiculite (me-

dium size) (2:1, v/v) and grown in insect-free climate chambers with 16 h

of light (100mE intensity) at 218C and 75% humidity.

Accession Numbers

Accession number for ESTs can be found in Table 1; amino acid

sequences in FASTA formatused forthe constructionof thephylogenetic

tree canbe found as Supplemental Data 1 onlineand at http://www.p450.

kvl.dk/Review2006/Figure1_Sequence_FASTA.tfa.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Positions of T-DNA Insertions in dex2-1and

dex2-2.

Supplemental Figure 2. GC-MS Analyses of Metabolites Produced

by CYP703A2 on Capric, Lauric, and Myristic Acids.

Supplemental Data 1.FASTA File of Amino Acid Sequences Used forthe Construction of the Phylogenetic Tree.

Supplemental Data 2. Multiple Sequence Alignment Used for the

Construction of the Phylogenetic Tree.

ACKNOWLEDGMENTS

We thank Jonathan Jones at the Sainsbury Laboratory (John Innes

Center, Norwich UK) for providing the SLAT T-DNA insertion line,

Joseph R. Ecker and the Salk Institute Genomic Analysis Laboratory

for providing the sequence-indexed T-DNA insertion mutant, and the

ABRC for distributing the seeds. We also thank Kerstin Brismar and

Salla Martina for assistance in scanning electron microscopy; Carl Erik

Olsen for LC-MS analyses; Christina Lunde for helpful discussions onP.

Patens; Suzanne M. Paquette for help with bioinformatics and for data

mining the poplar genome for CYP703orthologs; and finally Julia Hosp,

Ron Coolbaugh, and Peter Hedden for valuable scientific discussions.

Received July 15, 2006; revised April 1, 2007; accepted April 18, 2007;

published May 11, 2007.

REFERENCES

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DOI 10.1105/tpc.106.045948; originally published online May 11, 2007;2007;19;1473-1487Plant Cell

Werck-Reichhart and Sren BakMarc Morant, Kirsten Jrgensen, Hubert Schaller, Franck Pinot, Birger Lindberg Mller, Danile

Lauric Acid to Provide Building Blocks for Sporopollenin Synthesis in PollenCYP703 Is an Ancient Cytochrome P450 in Land Plants Catalyzing in-Chain Hydroxylation of

This information is current as of October 1, 2014

Supplemental Data http://www.plantcell.org/content/suppl/2007/05/10/tpc.106.045948.DC1.html

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